The production of lipid second messengers is stimulated by a variety of hormones and neurotransmitters that work through G protein coupled receptors (GPCR). One such lipid signaling system involves the hydrolysis of phosphatidylcholine, a major component of biological membranes, by phospholipase D (PLD) to produce phosphatidic acid (PA) and choline. Phosphatidic acid and its metabolic products, diacylglycerol and lyso-PA, have been postulated to modulate cell proliferation, vesicle transport membrane remodeling, long term goal of these studies is to achieve a better understanding of the molecular basis of PLD1 regulation. We decided to focus on the role of CDC42 in GPCR activation pathways because our preliminary findings suggests that Cdc42 modulates activation by other regulatory proteins and recent reports have shown that some heterotrimeric G proteins crosstalk to guanine nucleotide exchange factors specific to the Rho subfamily. The initial aim focuses on identifying sites on Cdc42 that bind and activate PLD1. We predict that several factors will be involved in heptahelical receptor activation of PLD1 and studies in the second aim will characterize two potentially novel modulators of PLD1 to determine their participation. The third aim will determine whether a purinergic receptor stimulation of PLD1 is transduced through Cdc42. The role of Cdc42 has not previously been systematically investigated. The proposed studies combine pharmacological, biochemical, and molecular genetic approaches to elucidate the molecular mechanisms of PLD1 activation in native cell lines. These studies will utilize both intact cells and cell-free preparations that retain purinergic receptor-stimulated activation of PLD1.